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Pasteurella haemolytica is a gram-negative bacterium that causes bovine shipping fever. The major virulence factor produced by P. haemolytica is a leukotoxin that lyses macrophages. While studying the transcriptional regulation of the P. haemolytica leukotoxin gene, it was observed that the leukotoxin (lk!) promoter is poorly expressed in Escherichia coli. This indicated that there may be trans-activators in P. haemolytica that are missing in the E. coli system. Three sequences upstream of the leukotoxin promoter share homology with other bacterial upstream activation sequences (UAS), and two of these are bound by P. haemolytica proteins in crude extracts. Using an Ikt-lacZ reporter gene fusion, a P. haemolytica cosmid library was screened to identify members that could activate leukotoxin transcription. Two cosmids, pSH250 and pSH2195, encoded products that bound to the UAS sequences and increased 𝛽-galactosidase activity of the Ikt-lacZ reporter fusion. My aim was to create restriction maps of pSH250 and pSH2195 and then to subclone fragments of these cosmids into the smaller vector pSC KS+ to identify the regions of each cosmid that encode putative activator function. For pSH250, ten fragments were subcloned, encompassing approximately 70% of the P. haemolytica insert. The resulting plasmids were transformed into the Ikt-lacZ fusion strain and 𝛽-galactosidase activity assays were performed . Of those assayed , 2 subcloned plasmids increased 𝛽-galactosidase activity two-fold and another increased the activity six-fold. Attempts to subclone a 6 kb portion of the pSH250 insert using different combinations of restriction enzymes have
been unsuccessful. A restriction map of pSH2195 has also been constructed
and one 1.5 kb fragment has been subcloned. Its activity has not yet been characterized. Future experiments will focus on characterization of those subclones that increase activity five-fold or more, using both 𝛽-galactosidase and DNA binding assays.